CN108795965A - A kind of screening technique of polar amino acid superior strain - Google Patents
A kind of screening technique of polar amino acid superior strain Download PDFInfo
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Abstract
The invention discloses a kind of screening techniques of screening polar amino acid superior strain.Screening technique provided by the present invention be according to include the following steps carry out：Determine that polar amino acid corresponds to codon usage frequency in the microorganism screened, select to express the gene of new resistance or phenotypic character in sieved bacterial strain as selection markers, all codons of a certain polar amino acid in its nucleic acid sequence are replaced with into rare codon, artificial synthesized screening-gene is simultaneously connected on screening plasmid.It is transformed into screening plasmid in the mutation library that the methods of induced mutations obtains microorganism, by detecting the expression of screening-gene, the bacterial strain of high yield polar amino acid can be picked out from mutant strain.The present invention, to the Preference of codon, realizes the screening to high yield polar amino acid bacterial strain, and successfully filter out the mutant strain of high yield L-arginine and Serine respectively in Escherichia coli using biology.
Description
Technical field
The present invention relates to a kind of screening techniques of polar amino acid superior strain, belong to technical field of bioengineering.
Background technology
Polar amino acid is the class of amino acid marked off according to side-chain radical polarity, including L- glycine, L- ammonia
Acid, L-threonine, L-cysteine, l-tyrosine, altheine, L-Glutamine, L-arginine, L-lysine, L- groups
Propylhomoserin, L-Aspartic acid and Pidolidone, also known as hydrophilic amino acid.Polar amino acid has widely in terms of food and medicine
Using L-threonine can relieve fatigue, enhancing development；L-cysteine is added in fruit juice to prevent vitamin C from aoxidizing
And brown stain；L-lysine is one of human body ketogenic amino acid, can promote calcium uptake, improves sleep, improves memory, is first
Limiting amino acid.
Currently, microbial fermentation production polar amino acid still has large stretch of blank, there is an urgent need to the outstanding amino acid of character is high
Produce bacterial strain.Traditional screening technique is to carry out repeated screening using amino acid analogue to obtain, such as Serine and L- essence ammonia
The corresponding hydroxamic acid of acid can be used as its analog, the superior strain for screening Serine and L-arginine.But according to
Rely the screening mode of amino acid analogue that can be limited to the shadow of amino acid analogue type, turn-over capacity and extracellular row's ability
It rings, must take turns repetition screening can just obtain real amino acid superior strain more.
The screening that amino acid superior strain is carried out using codon possesses many advantages.First, with gene substitution amino acid
Analog, for different plant species optimization, the species quantity that can be screened is much larger than the scope of application of amino acid analogue.Its
Secondary, rare codon acts only in the gene containing this codon the screening pressure of amino acid superior strain, will not interfere
To the normal growth of the expression and influence thalline of other genes；And amino acid analogue not only can in radom insertion to peptide chain, and
And the biosynthesis of purine, pyrimidine and ATP can be inhibited, there is apparent toxic effect to thalline.
Invention content
The purpose of the present invention is to provide a kind of from nature or artificial mutation library screens the side of amino acid superior strain
Method is simply and effectively isolated from numerous bacterial strains corresponding using the difference of codon preference and tRNA aminoacylations
Amino acid superior strain tests the final amino acid production for determining bacterial strain eventually by shake flask fermentation, is amino acid superior strain
Screening mode new method (such as Fig. 1) is provided.
According to technical solution provided by the invention, a kind of screening technique of amino acid superior strain is walked using following methods
Suddenly：
1. analyzing Escherichia coli (Escherichia coli) and corynebacterium glutamicum (Corynebacterium
Glutamicum L- glycine in), Serine, L-threonine, L-cysteine, l-tyrosine, altheine, L- paddy ammonia
The codon preference of amide, L-arginine, L-lysine, L-Histidine, L-Aspartic acid and Pidolidone, according to what is screened
Amino acid and strain select corresponding rare codon.
2. determining screening-gene, and a kind of codon of polar amino acid in screening-gene is replaced in step 1 and is determined
Rare codon, and this artificial synthesized sequence.
3. screening-gene sequence artificial synthesized in step 2 is connect with plasmid vector, connection product is gone into large intestine
It in bacillus, is verified by PCR, selects correct single bacterium colony, extraction obtains screening plasmid.
4. the bacterial strain screened is prepared competence, the plasmid obtained in step 3 is gone into bacterial strain competence using electricity
In, it is applied on tablet and cultivates.
5. the single bacterium colony converted in step 4 is transferred in screening and culturing medium one by one, cultivated using 96 orifice plates
- 24 hours 10 hours or 10 hours or 16 hours or 24 hours.By the expression quantity or picking thalline feature that measure screening-gene
Most apparent bacterial strain is as amino acid superior strain.
6. obtained amino acid superior strain is carried out fermentation verification in the fermentation medium.
Screening and culturing medium forms：Tryptone 0.5-20g/L, yeast powder 0.5-20g/L, sodium chloride 0.5-20g/L.
Fermentation medium forms：Phosphate 5-20g/L, ammonium chloride 1-5g/L, glucose 1-5g/L, sodium chloride 0.1-
10g/L, magnesium sulfate 0.3g/L, calcium chloride 0.015g/L, vitamin B15-50μg/L.
Description of the drawings
Fig. 1 is the principle that polar amino acid superior strain is screened using rare codon；
Fig. 2 is the fermentation results of the L-arginine superior strain filtered out using rare codon；
Fig. 3 is the fermentation results of the Serine superior strain sifted out using rare codon.
Specific implementation mode
Experimental method used in following examples is conventional method unless otherwise specified.
Material, reagent etc. used in following examples unless otherwise specified can be from acquisitions of commercially advancing by leaps and bounds.
The following examples are further illustrations of the invention, the limitation to substantive content of the present invention is not constituted.
Embodiment 1 screens L-arginine superior strain using rare codon
(1) with bacillus coli DH 5 alpha (being purchased from Beijing Bo Maide gene technology Co., Ltd) for bacterium, Escherichia coli
The L-arginine codon that DH5 α are used in translation process shares six kinds, is CGG, CGU, CGA, CGC, AGG and AGA respectively,
The frequency of use of wherein codon AGG it is minimum (non-patent literature for recording e. coli codon Preference is Dong H,
Nilsson L, Kurland C G., 1996, Co-variation of tRNA abundance and codon usage in
Escherichia coli at different growth rates.Journal of molecular biology,260
(5):649-663.), therefore it can be used for arginine-producing strain screening.By 8 arginine pair in kanamycin gene Kan
The codon answered all replaces with rare codon AGG.Kanamycins shown in sequence 1 is obtained using the method for total gene synthesis
Gene.
(2) total gene synthesis system is：5×FastPfu Fly Buffer 10 μ L, dNTP (2.5mM) 4 μ
L, 2 μ L of primer premixed liquid,FastPfu Fly DNA Polymerase 1 μ L, 35 μ L of distilled water, total volume 50
μL.Amplification condition is 95 DEG C and is denaturalized anneal within 20 seconds, 55 DEG C 20 seconds, 72 DEG C of extensions 20 seconds (30 cycles)；72 DEG C extend 5 minutes (1
A cycle).Amplified production includes kanamycin gene Kan, and is connected on pAKR carrier frameworks.Linked system：1.5μL
Pcr amplification product, 1 μ L pAKR carrier segments, 7.5 μ L GibsonMaster Mix (are purchased from NEW ENGLAND
BioLabs), it is gently mixed, 50 DEG C of water-baths 60 minutes.It is then added in 50 μ L DH5 α competent cells and (it is rich to be purchased from Beijing
Mai De gene technology Co., Ltd), ice bath 30 minutes, 42 DEG C of heat shocks 30 seconds are placed in 2 minutes on ice at once.250 μ L SOC are added
Culture medium, the shake culture 1 hour in 37 DEG C, 200rpm shaking tables.Because pAKR skeletons contain ampicillin resistance gene
Amp takes 200 μ L bacterium solutions to be applied on the LB tablets containing ampicillin, is incubated overnight.After growing single bacterium colony on LB tablets,
Positive colony is subjected to Liquid Culture after PCR sequence verifications, extraction plasmid carries out sequence verification.Sequencing result shows carrier
PAKR has been successively inserted into newly synthesized kanamycin gene Kan, it was demonstrated that plasmid construction is correct, and obtained recombinant plasmid is named as
pAKR-RC。
Mutagenic treatment is carried out to DH5 α by ultraviolet, nitrosoguanidine or normal temperature and pressure plasma mutagenesis system, will be handled
Bacterium solution afterwards is forwarded in 2mL LB culture mediums.3-5 hours are cultivated until OD through 37 DEG C, 200rpm600To 0.4-0.6, by bacterium
Liquid is placed in 15-30 minutes on ice, is then centrifuged 10 minutes, is discarded supernatant at 4 DEG C, 4000 × g, with the sterile ice water weights of 500 μ L
Outstanding cell is centrifuged 10 minutes in 4 DEG C, 4000 × g, is carefully discarded supernatant again.Cell is resuspended to 50 μ L with 20% glycerite.
Electricity turns condition：The 50 μ L competent cells prepared are placed on ice, 1 μ L carrier pAKR-RC are added, are placed 10 minutes on ice
Afterwards, 0.2cm Bio-Red electricity revolving cups are gone to.Use Gene Pulser XcellTMElectric perforating system (Bio-Red companies), electric shock
Voltage is 2.5kV.Electric revolving cup is rinsed with 500 μ L LB culture mediums, be transferred in test tube after slight pressure-vaccum 5 times, 37 at once after electric shock
DEG C, 200rpm cultivate half an hour, bacterium solution is applied on the LB tablets containing kanamycins and ampicillin, 37 DEG C are incubated overnight
Afterwards, the mutant bacteria library containing pAKR-RC plasmids is obtained.
The picking monoclonal from tablet is forwarded in 96 deep-well plates containing 1.5mL screening and culturing mediums, 37 DEG C, 200rpm
After culture 12-24 hours, the light absorption value of bacterium solution is measured at 600nm wavelength, selects the highest 10 plants of bacterium of light absorption value as possible
L-arginine superior strain.
Screening and culturing medium forms：Tryptone 2g/L, yeast powder 1g/L, sodium chloride 2g/L, 50 μ g/mL of kanamycins.
Embodiment 2 is screened to obtain mutant strain fermenting and producing L-arginine with rare codon
The highest 10 plants of Escherichia coli of light absorption value filtered out from embodiment 1, picking single bacterium colony are seeded to seed culture medium
In.Seed culture medium forms：Tryptone 10g/L, yeast powder 5g/L, sodium chloride 5g/L, 50 μ g/ml of kanamycins, culture medium
pH7.0。
Seed culture medium is 10ml in 50ml triangular flasks, and 121 DEG C sterilize 20 minutes.It is prominent that 10 plants of Escherichia coli are inoculated with after cooling
Become bacterium and DH5 α bacterial strains, cultivation temperature is 37 DEG C, shaking speed 200rpm, after cultivating 12 hours, measures the suction of all bacterium solutions
Light value.Thalline were collected by centrifugation by 8000 × g, and using sterile water wash 3 times, the light absorption value for adjusting all seed liquors is identical, for sending out
Ferment culture medium inoculated.
In 250ml triangular flasks fermentation medium be 20ml, 115 DEG C sterilizing 20min after.Inoculum concentration is 0.1% (v/v), hair
Ferment temperature is 37 DEG C, shaking speed 250rpm, and fermentation time is 24 hours.
Analysis method：The L-Leu in zymotic fluid is measured using Shimadzu high performance liquid chromatograph.Zymotic fluid is logical
Phenyl isothiocyanate derivation process is crossed, carries out separation and quantitative using the silent winged C18 chromatographic columns of match later.As a result display shares 7 plants and dashes forward
The L-arginine yield of mutant, which is higher than control strain DH5 α, control strain DH5 every gram of thalline of α, can generate 0.28mg L- essences
Propylhomoserin, mutant strain RP-5 can reach every gram of thalline and generate 0.69mg L-arginines, and 2.46 times are improved compared to control strain DH5 α
(such as Fig. 2).
Embodiment 3 screens Serine superior strain using rare codon
With bacillus coli DH 5 alpha (being purchased from Beijing Bo Maide gene technology Co., Ltd) for bacterium, bacillus coli DH 5 alpha
The Serine codon used in translation process shares six kinds, is UCU, UCC, UCG, UCA, AGU and AGC respectively.For
Escherichia coli, frequencies of the codon UCC in genome it is relatively low (record e. coli codon Preference non-patent literature be
Dong H, Nilsson L, Kurland C G., 1996, Co-variation of tRNA abundance and codon
usage in Escherichia coli at different growth rates.Journal of molecular
biology,260(5):649-663.), therefore it can be used for serine Screening of strain with high productivity.It will be in spectinomycin gene Spec
The corresponding codon of 17 serines all replace with codon UCC.It is obtained shown in sequence 2 using the method for total gene synthesis
Spectinomycin gene Spec.
Total gene synthesis system is：5×FastPfu Fly Buffer 10 μ L, dNTP (2.5mM) 4 μ L,
2 μ L of primer premixed liquid,1 μ L of FastPfu Fly DNA Polymerase, 35 μ L of distilled water, total volume is 50 μ
L.Amplification condition is 95 DEG C and is denaturalized anneal within 20 seconds, 55 DEG C 20 seconds, 72 DEG C of extensions 20 seconds (30 cycles)；72 DEG C extend 5 minutes (1
A cycle).Amplified production includes aminoglycoside adenylyl transferase gene and its each 200 bases of upstream and downstream, and is connected
Onto pAKR carrier frameworks.Linked system：1.5 μ L pcr amplification products, 1 μ L pGFP carrier segments, 7.5 μ L GibsonMaster Mix (be purchased from NEW ENGLAND BioLabs), are gently mixed, 50 DEG C of water-baths 60 minutes.Then
It is added in 50 μ L DH5 α competent cells and (is purchased from Beijing Bo Maide gene technology Co., Ltd), ice bath 30 minutes, 42 DEG C of heat shocks
It 30 seconds, is placed at once 2 minutes on ice.250 μ L SOC culture mediums, the shake culture 1 hour in 37 DEG C, 200rpm shaking tables is added.
It takes 200 μ L bacterium solutions to be applied on the LB tablets containing spectinomycin, is incubated overnight.After growing single bacterium colony on LB tablets, surveyed through PCR
Positive colony is subjected to Liquid Culture after sequence verification, extraction plasmid carries out sequence verification.Sequencing result shows carrier pSSer successes
Insert newly synthesized spectinomycin gene Spec, it was demonstrated that plasmid construction is correct, and obtained recombinant plasmid is named as pSSer-
RC。
Mutagenic treatment is carried out to DH5 α by ultraviolet, nitrosoguanidine or normal temperature and pressure plasma mutagenesis system, will be handled
Bacterium solution afterwards is forwarded in 2mL LB culture mediums.3-5 hours are cultivated until OD through 37 DEG C, 200rpm600To 0.4-0.6, by bacterium
Liquid is placed in 15-30 minutes on ice, is then centrifuged 10 minutes, is discarded supernatant at 4 DEG C, 4000 × g, with the sterile ice water weights of 500 μ L
Outstanding cell is centrifuged 10 minutes in 4 DEG C, 4000 × g, is carefully discarded supernatant again.Cell is resuspended to 50 μ L with 20% glycerite.
Electricity turns condition：The 50 μ L competent cells prepared are placed on ice, 1 μ L carrier pSSer-RC are added, in 10 points of placement on ice
Zhong Hou goes to 0.2em Bio-Red electricity revolving cups.Use Gene Pulser XcellTMElectric perforating system (Bio-Red companies), electricity
It is 2.5kV to hit voltage.Electric revolving cup is rinsed with the LB culture mediums of 500 μ L, test tube is transferred to after slight pressure-vaccum 5 times at once after electric shock
In, 37 DEG C, 200rpm culture half an hour, bacterium solution is applied on the LB tablets containing spectinomycin, after 37 DEG C are incubated overnight, is obtained
Mutant bacteria library containing pSSer-RC plasmids.
The picking monoclonal from tablet is forwarded in 96 deep-well plates containing 1.5mL screening and culturing mediums, 37 DEG C, 200rpm
After culture 12-24 hours, the light absorption value of bacterium solution is measured at 600nm wavelength, selects the highest 10 plants of bacterium of light absorption value as possible
Serine superior strain.
Screening and culturing medium group becomes：Tryptone 2g/L, yeast powder 1g/L, sodium chloride 2g/L, 25 μ g/mL of spectinomycin.
Embodiment 4 is screened to obtain mutant strain fermenting and producing Serine with rare codon
The highest 10 plants of Escherichia coli of light absorption value filtered out from embodiment 3, picking single bacterium colony are seeded to seed culture medium
In.Seed culture medium forms：Tryptone 10g/L, yeast powder 5g/L, sodium chloride 5g/L, 50 μ g/ml of spectinomycin, culture medium
pH7.0。
Seed culture medium is 10ml in 50ml triangular flasks, and 121 DEG C sterilize 20 minutes.It is prominent that 10 plants of Escherichia coli are inoculated with after cooling
Become bacterium and DH5 α bacterial strains, cultivation temperature is 37 DEG C, shaking speed 200rpm, after cultivating 12 hours, measures the suction of all bacterium solutions
Light value.Thalline were collected by centrifugation by 8000 × g, and using sterile water wash 3 times, the light absorption value for adjusting all seed liquors is identical, for sending out
Ferment culture medium inoculated.
In 250ml triangular flasks fermentation medium be 20ml, 115 DEG C sterilizing 20min after.Inoculum concentration is 0.1% (v/v), hair
Ferment temperature is 37 DEG C, shaking speed 250rpm, and fermentation time is 24 hours.
Analysis method：The Serine in zymotic fluid is measured using Shimadzu high performance liquid chromatograph.Zymotic fluid is logical
Phenyl isothiocyanate derivation process is crossed, carries out separation and quantitative using using the silent winged C18 chromatographic columns of match later.Control strain DH5 α are every
Gram thalline can generate 0.07mg Serines, and mutant strain SP-2 can reach every gram of thalline and generate 0.53mg L-arginines,
7.57 times (such as Fig. 3) are improved compared to control strain DH5 α.
Sequence table
<110>Beijing Institute of Technology
<120>A kind of screening technique of polar amino acid superior strain
<150> 201810180437.6
<151> 2018-03-05
<160> 2
<170> SIPOSequenceListing 1.0
<210> 2
<211> 816
<212> DNA
<213>Escherichia coli (Escherichia coli)
<400> 2
atgagccata ttcaacggga aacgtcttgc tctaggccgc gattaaattc caacatggat 60
gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc 120
tatcgattgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc 180
gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct 240
cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg 300
atccccggga aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt 360
gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct 420
tttaacagcg atagggtatt taggctcgct caggcgcaat caaggatgaa taacggtttg 480
gttgatgcga gtgattttga tgacgagagg aatggctggc ctgttgaaca agtctggaaa 540
gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca 600
cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggaagggtc 660
ggaatcgcag acaggtacca ggatcttgcc atcctatgga actgcctcgg tgagttttct 720
ccttcattac agaaaaggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa 780
ttgcagtttc atttgatgct cgatgagttt ttctaa 816
<210> 2
<211> 942
<212> DNA
<213>Escherichia coli (Escherichia coli)
<400> 2
atggcttgtt atgactgttt ttttggggta cagtccatgc ctcgggcatc caagcagcaa 60
gcgcgttacg ccgtgggtcg atgtttgatg ttatggtcct ccaacgatgt tacgcagcag 120
ggctcccgcc ctaaaacaaa gttaaacatc atgagggaag cggtgatcgc cgaagtatcc 180
actcaactat ccgaggtagt tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc 240
gtacatttgt acggctccgc agtggatggc ggcctgaagc cacactccga tattgatttg 300
ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc gagctttgat caacgacctt 360
ttggaaactt ccgcttcccc tggagagtcc gagattctcc gcgctgtaga agtcaccatt 420
gttgtgcacg acgacatcat tccgtggcgt tatccagcta agcgcgaact gcaatttgga 480
gaatggcagc gcaatgacat tcttgcaggt atcttcgagc cagccacgat cgacattgat 540
ctggctatct tgctgacaaa agcaagagaa cattccgttg ccttggtagg tccagcggcg 600
gaggaactct ttgatccggt tcctgaacag gatctatttg aggcgctaaa tgaaacctta 660
acgctatgga actccccgcc cgactgggct ggcgatgagc gaaatgtagt gcttacgttg 720
tcccgcattt ggtactccgc agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac 780
tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca tacttgaagc tagacaggct 840
tatcttggac aagaagaaga tcgcttggcc tcccgcgcag atcagttgga agaatttgtc 900
cactacgtga aaggcgagat caccaaggta gtcggcaaat aa 942
Claims (5)
1. a kind of method for screening polar amino acid superior strain using rare codon, it is characterized in that analysis microorganism is to password
The Preference of son realizes the screening to polar amino acid superior strain by importing the foreign gene replaced through rare codon
Effect.
2. according to a kind of method for screening amino acid superior strain using rare codon described in claim, it is characterised in that adopt
Use following steps：
A. analysis microbial strains to glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,
The Preference of arginine, lysine, histidine, aspartic acid and glutamic acid histidine codon determines that specific its translates polarity ammonia
The corresponding rare codon of base acid；
B. according to the growth characteristics of microbial strains, select suitable screening-gene, including but not limited to antibiotics resistance gene,
The gene of detectable fluorescin, the lethal gene for causing thalline death and influence colony colour, form；
C. according to sieved microbial strains to the Preference of amino acid codes, by glycine, serine, Soviet Union's ammonia in screening-gene
Acid, cysteine, tyrosine, asparagine, glutamine, arginine, lysine, histidine, aspartic acid or glutamic acid ammonia
The codon of acid replaces with corresponding rare codon, and artificial synthesized screening-gene sequence.
D. screening-gene sequence artificial synthesized in step C is connect with plasmid vector, obtains the plasmid for screening, and convert
Into the mutation library of sieved bacterial strain；
E. the single bacterium colony that conversion obtains is transferred in screening and culturing medium one by one, incubation time is -24 hours 10 hours.Pass through survey
Determine screening-gene expression quantity or the most apparent bacterial strain of picking thalline feature as amino acid superior strain；
F. the polar amino acid superior strain for screening acquisition in step E is seeded to fermentation medium fermentation, measures Polar Amides
The yield of acid.
3. a kind of method for screening polar amino acid superior strain using rare codon as claimed in claim 1 or 2, special
Sign is in step (A) that the rare codon is any codon that can be inserted into amino acid in peptide chain, and described is micro-
Biological bacterial strain includes but not limited to all microorganisms that polar amino acid is synthesized in intracellular such as Escherichia coli, bar bacterium.
4. a kind of method for screening polar amino acid superior strain using rare codon as claimed in claim 1 or 2, special
Sign is in step (B), it is described for screening-gene include fluorescence, antibiotic resistance, lethal gene or change thalline color,
The gene of form.
5. a kind of method for screening amino acid superior strain using rare codon as claimed in claim 1 or 2, feature exist
In step (D), the composition of screening and culturing medium includes tryptone 0.5-20g/L, yeast powder 0.5-10g/L, sodium chloride 0.5-
20g/L, cultivation temperature are 30-42 DEG C, pH 6.0-8.0.
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| CN110607317A (en) * | 2019-09-27 | 2019-12-24 | 北京理工大学 | Method for regulating gene expression by using novel dCas9 |
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