CN108795966A - A kind of screening technique of branched-chain amino acid superior strain - Google Patents
A kind of screening technique of branched-chain amino acid superior strain Download PDFInfo
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Abstract
The present invention provides a kind of screening technique of branched-chain amino acid superior strain.Screening technique provided by the present invention be according to include the following steps carry out：Determine that branched-chain 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 branched-chain 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 branched-chain amino acid can be picked out from mutant strain.The bacterial strain that method provided by the present invention can be directed to different microorganisms bacterial screening high yield branched-chain amino acid can be used for the bacterial strain of high flux screening high yield branched-chain amino acid without selecting respective amino acid analogue.
Description
Technical field
The present invention relates to a kind of screening techniques of branched-chain amino acid superior strain, belong to technical field of bioengineering.
Background technology
Branched-chain amino acid refers to the class of amino acid for having aliphatic lateral chain, can be in the natural amino acid of synthetic protein
Including three kinds of L-Leu, l-Isoleucine and Valine amino acid.Branched-chain amino acid belongs to 8 kinds of amino needed by human
Acid accounts for 40% or so of mammal essential amino acid.Branched-chain amino acid is in metabolism and physiologically has very important effect.Branch
Chain amino acid generally increases the anabolism function such as muscle growth by promoting the release of insulin and growth hormone.In addition,
Branched-chain amino acid shares same transhipment egg with other aromatic amino acids (such as L-Trp, l-tyrosine and L-phenylalanine)
Enter brain in vain, can both can be used for energizing with the synthesis of regulatory protein matter and neurotransmitter in the brain.With 70kg at
Year artificial example is respectively 2.9g, 1.3g and 0.3g per the amount of L-Leu, l-Isoleucine and Valine is taken in day by day.
There are corynebacterium glutamicum (Corynebacterium glutamicum) and Escherichia coli at present
(Escherichia coli) be used for branched-chain amino acid fermenting and producing, these mutant strains mostly be using amino acid analogue into
The more wheel repeated screenings of row obtain.Such as norvaline (norvaline), 4- nitrogen-leucine (4-azaleucine) and 2- amino -3-
(methyl mercapto) butyric acid (4-thiaisoleucine) has been respectively used to Valine, L-Leu and l-Isoleucine Producing Strain
The screening of strain.But it is not that the analog of any branched-chain amino acid can be used for this screening technique, for example be equally different bright
The analog of propylhomoserin, 4- nitrogen-isoleucine (4-azaisoleucine) cannot be used for this screening technique, this with regard to increasing on foot
Find the work of amino acid analogue.Amino acid analogue type, micro- can be limited to by relying on the screening mode of amino acid analogue
The influence of biotransport ability and extracellular row's ability, the more wheels of palpus, which repeat screening, can just obtain real amino acid superior strain.
Rare codon is a kind of intrinsic but special codon of inter-species of biology of generation during biological evolution, for not
Corresponding rare codon can be determined according to codon usage frequency with biology, different aminoacids.Therefore, it will be substituted dilute
There is the exogenous gene sequence of codon to be connected on plasmid and be transformed into purpose microorganism, the translation skill of foreign gene relies on
In the concentration of intracellular amino acid, by selecting the different foreign genes such as resistance, fluorescence or chromoprotein, intracellular amino acid concentration
Higher microorganism can be realized according to growth vigor, fluorescence intensity or form and screen.In addition, rare codon is to amino acid height
The screening pressure of production bacterial strain acts only in the gene containing this codon, will not interfere and influence phage gene group gene just
Often expression.Therefore, there is more wide application prospect using the screening technique of rare codon.
Invention content
The purpose of the present invention is to provide one kind screening branched-chain amino acid high yield from nature or artificial mutation microorganism
The method of bacterial strain is efficiently isolated from microbial strains using the difference of codon preference and tRNA aminoacylations
Corresponding branched-chain amino acid superior strain tests the final amino acid production for determining bacterial strain eventually by shake flask fermentation, is branch
The screening mode of amino acid superior strain provides new method (such as Fig. 1).
According to technical solution provided by the invention, a kind of screening technique of branched-chain amino acid superior strain is used with lower section
Method step：
1. analyzing Escherichia coli (Escherichia coli) and corynebacterium glutamicum (Corynebacterium
Glutamicum the codon preference of Valine in), L-Leu and l-Isoleucine, according to the amino acid that is screened and
Strain selects corresponding rare codon.
2. determining screening-gene, and the amino acid codes of valine, leucine or isoleucine in screening-gene are replaced
It is changed to the rare codon determined in step 1, and artificial synthesized screening-gene 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 used into conversion to bacterial strain competence
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, 96 deep-well plates cultures 10 are used
- 24 hours or 10 hours or 16 hours or 24 hours hour.By measuring expression quantity or sight of the screening-gene in different strains
Morphological features are examined, select most apparent bacterial strain as amino acid superior strain.
Screening and culturing medium group becomes：Tryptone 0.5-20g/L, yeast powder 0.5-20g/L, sodium chloride 0.5-20g/L,
pH6.0-8.0。
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 B1 5-50 μ g/L, pH6.0-8.0.
Description of the drawings
Fig. 1 is the principle that branched-chain amino acid Producing Strain is screened using rare codon；
Fig. 2 is the fermentation results that L-Leu superior strain is filtered out using rare codon.
Fig. 3 is the fermentation results that Valine superior strain is filtered 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-Leu 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 leucine codons that DH5 α translations use share six kinds of UUG, UUA, CUC, CUG, CUU and CUA, and wherein codon CUA's makes
With frequency is minimum (non-patent literature of e. coli codon Preference is recorded as 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.).It is mould that will be blocked
Corresponding 29 leucine codons of leucine all replace with leucine rare codon CUA in plain gene Kan nucleic acid sequences,
Kanamycin gene shown in sequence 1 is obtained using the method for total gene synthesis.
(2) total gene synthesis system is：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 is kanamycin gene, and is connected on pKan carriers.Linked system：1.5 μ L PCR amplifications
Product, 1 μ L pKan carrier segments, 7.5 μ L GibsonMaster Mix (are purchased from NEW ENGLAND
BioLabs), it is gently mixed, 50 DEG C of water-baths 30-60 minutes.It is then added in 50 μ L DH5 α competent cells and (is purchased from north
Jing Bomaide gene technology Co., Ltd), ice bath 30 minutes, 42 DEG C of heat shocks 60 seconds are placed in 2 minutes on ice at once.250 μ L are added
SOC culture mediums, the shake culture 1 hour in 37 DEG C, 200rpm shaking tables.200 μ L bacterium solutions are taken to be applied to the LB containing kanamycins flat
On plate, after being incubated overnight, positive colony be transferred in LB liquid medium by 5 single bacterium colonies of picking after PCR sequence verifications
37 DEG C, 200rpm be incubated overnight, extraction plasmid carry out sequence verification.Sequencing result shows to insert in carrier pKan newly synthesized
Kanamycin gene, it was demonstrated that plasmid construction is correct, and obtained recombinant plasmid is named as pKan-RC.
(3) mutagenic treatment is carried out to DH5 α bacterial strains by ultraviolet, nitrosoguanidine or normal temperature and pressure plasma mutagenesis system,
By treated, bacterium solution is forwarded in 2ml LB culture mediums.3-5 hours are cultivated until light absorption value to 0.4- through 37 DEG C, 200rpm
0.6, bacterium solution is placed in 15-30 minutes on ice, then centrifuges 10 minutes, discards supernatant at 4 DEG C, 4000 × g, with 500 μ L without
Cell is resuspended in bacterium water, centrifuges 10 minutes in 4 DEG C, 4000 × g, carefully discards supernatant again.Cell is resuspended with 20% glycerite
To 50 μ L.Electricity turns condition：The 50 μ L competent cells prepared are placed on ice, 1 μ L carrier pKan-RC are added, are put on ice
It sets after ten minutes, goes to 0.2cm Bio-Red electricity revolving cups.Use Gene Pulser XcellTM(Bio-Red is public for electric perforating system
Department), shock voltage 2.5kV.Electric revolving cup is rinsed with the LB culture mediums of 500 μ L, be transferred to after slight pressure-vaccum 5 times at once after electric shock
In test tube, 37 DEG C, 200rpm culture half an hour, bacterium solution is applied on the LB tablets containing kanamycins, after 37 DEG C are incubated overnight,
Obtain the mutation flora containing pKan-RC plasmids.
(4) the picking monoclonal from tablet is forwarded in 96 deep-well plates containing 1.5ml screening and culturing mediums, 37 DEG C,
After 200rpm is cultivated 12 hours, measurement and thalline light absorption value select the highest bacterial strain of light absorption value as possible L-Leu height
Produce bacterial strain.
Screening and culturing medium forms：Tryptone 5g/L, yeast powder 1g/L, sodium chloride 5g/L, 50 μ g/ml of kanamycins, training
Support base pH7.0.
Embodiment 2 is screened to obtain mutant strain fermenting and producing L-Leu 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 10g/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.L-Leu
It is quantitative handle zymotic fluid using phenyl isothiocyanate derivatization method, separation and quantitative is carried out using the silent winged C18 chromatographic columns of match.As a result it shows
Show that the L-Leu yield that the L-Leu yield for sharing 8 plant mutant strains is higher than control strain DH5 α, control strain DH5 α is
0.491g/L, the L-Leu yield of LM-1 can reach 1.942g/L in sieved mutant strain, be improved compared to control strain DH5 α
3.95 times (such as Fig. 2).
Embodiment 3 screens Valine superior strain using rare codon
(1) with Escherichia coli Ser-19 (Laboratories Accession bacterium) for bacterium, Escherichia coli Ser-19 translations use
Valine codon shares tetra- kinds of GUU, GUC, GUA and GUG, and the frequency of use of wherein codon GUC is minimum (to record Escherichia coli
The non-patent literature of codon preference is Dong H, Ni lsson L, Kurland C G., 1996, Co-variation of
tRNA abundance and codon usage in Escherichia coli atdifferent growth
rates.Journal of molecular biology,260(5):649-663.).By kanamycin gene Kan nucleic acid sequences
Corresponding 16 codons of middle valine all replace with valine rare codon GUC, are obtained using the method for total gene synthesis
To the kanamycin gene for replacing with rare codon valine.
(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 are
50μL.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 cycle).Amplified production is kanamycin gene, and is connected on pKan carriers.Linked system：1.5 μ L PCR expand
Increase production object, 1 μ L pKan carrier segments, 7.5 μ L GibsonMaster Mix (are purchased from NEW ENGLAND
BioLabs), it is gently mixed, 50 DEG C of water-baths 30-60 minutes.The competence that 50 μ L Ser-19 mutation libraries make then is added
In cell (Ser-19 be Laboratories Accession bacterial strain), ice bath 20 minutes, 42 DEG C of heat shocks 90 seconds are placed in 2 minutes on ice at once.It is added
250 μ L SOC culture mediums, the shake culture 1 hour in 37 DEG C, 200rpm shaking tables.200 μ L bacterium solutions are taken to be applied to containing kanamycins
LB tablets on, after being incubated overnight, positive colony be transferred to the training of LB liquid by 5 single bacterium colonies of picking after PCR sequence verifications
Support base in 37 DEG C, 200rpm be incubated overnight, extraction plasmid carry out sequence verification.Sequencing result shows to insert in carrier pKan new
The kanamycin gene of synthesis, it was demonstrated that plasmid construction is correct, and obtained recombinant plasmid is named as pKan-RC.
(3) Ser-19 bacterial strains are carried out at mutagenesis by ultraviolet, nitrosoguanidine or normal temperature and pressure plasma mutagenesis system
Reason, by treated, bacterium solution is forwarded in 2ml LB culture mediums.3-5 hours are cultivated until light absorption value arrives through 37 DEG C, 200rpm
Bacterium solution is placed in 15-30 minutes on ice, then centrifuges 10 minutes, discard supernatant at 4 DEG C, 4000 × g, with 500 μ by 0.4-0.6
Cell is resuspended in L sterile waters, centrifuges 10 minutes in 4 DEG C, 4000 × g, carefully discards supernatant again.It is resuspended with 20% glycerite thin
Born of the same parents are to 50 μ L.Electricity turns condition：The 50 μ L competent cells prepared are placed on ice, 1 μ L carrier pKan-RC are added, on ice
It places after ten minutes, goes to 0.2cm Bio-Red electricity revolving cups.Use Gene Pulser XcellTMElectric perforating system (Bio-Red
Company), shock voltage 2.5kV.Electric revolving cup is rinsed with the LB culture mediums of 500 μ L, shifted after slight pressure-vaccum 5 times at once after electric shock
Into test tube, 37 DEG C, 200rpm culture half an hour, bacterium solution is applied on the LB tablets containing kanamycins, 37 DEG C are incubated overnight
Afterwards, the mutation flora containing pKan-RC plasmids is obtained.
(4) the picking monoclonal from tablet is forwarded in 96 deep-well plates containing 1.5ml screening and culturing mediums, 37 DEG C,
After 200rpm is cultivated 12 hours, measurement and thalline light absorption value select the highest bacterial strain of light absorption value as possible Valine height
Produce bacterial strain.
Screening and culturing medium forms：Tryptone 5g/L, yeast powder 1g/L, sodium chloride 5g/L, 50 μ g/ml of kanamycins, training
Support base pH7.0.
Embodiment 4 is screened to obtain mutant strain fermenting and producing Valine 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 10g/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 Ser-19 bacterial strains, cultivation temperature is 37 DEG C, shaking speed 200rpm, after cultivating 12 hours, measures all bacterium solutions
Light absorption 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, is used for
Fermentation medium is inoculated with.
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 Valine in zymotic fluid is measured using Shimadzu high performance liquid chromatograph.Valine
It is quantitative handle zymotic fluid using phenyl isothiocyanate derivatization method, separation and quantitative is carried out using the silent winged C18 chromatographic columns of match.As a result it shows
Show that the Valine yield for sharing 10 plant mutant strains is higher than control strain Ser-19, the Valine production of control strain Ser-19
It is 0.67mg/g to measure, and the Valine yield of V-7 can reach 2.69mg/g in sieved mutant strain, compare control strain Ser-19
It improves 4.02 times (such as Fig. 3).
Sequence table
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<120>A kind of screening technique of branched-chain amino acid superior strain
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<151> 2018-03-05
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atccccggga aaacagcatt ccaggtacta gaagaatatc ctgattcagg tgaaaatatt 360
gttgatgcgc tagcagtgtt cctacgccgg ctacattcga ttcctgtttg taattgtcct 420
tttaacagcg atcgcgtatt tcgtctagct caggcgcaat cacgaatgaa taacggtcta 480
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Claims (5)
1. a kind of method for screening branched-chain amino acid superior strain using rare codon, it is characterized in that analysis microorganism is to password
The Preference of son realizes the work of screening branched-chain amino acid superior strain by importing the foreign gene replaced through rare codon
With.
2. being existed according to a kind of method for screening branched-chain amino acid superior strain using rare codon, feature described in claim
In using following steps：
A. analysis microbial strains are to the Preference of leucine, isoleucine and valine codon, determine its translate leucine,
The rare codon of isoleucine and valine；
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, lethal gene and influence colony colour, form；
C. according to sieved microbial strains to the Preference of amino acid codes, by leucine in screening-gene, isoleucine or
The codon of valine 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 branched-chain amino acid superior strain；
F. the branched-chain amino acid superior strain for screening acquisition in step E is seeded to fermentation medium fermentation, measures branched-amino
The yield of acid.
3. a kind of method for screening branched-chain 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, described
Microbial strains include but are not limited to all microorganisms that branched-chain amino acid is synthesized in intracellular such as Escherichia coli, corynebacteria.
4. a kind of method for screening branched-chain 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 (E), the composition of the screening and culturing medium includes tryptone 0.5-20g/L, yeast powder 0.5-10g/L, chlorination
Sodium 0.5-20g/L, cultivation temperature are 30-42 DEG C, pH 6.0-8.0.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592126A (en) * | 2019-09-27 | 2019-12-20 | 北京理工大学 | Method for regulating gene expression at translation level by using rare codon |
| CN110607317A (en) * | 2019-09-27 | 2019-12-24 | 北京理工大学 | Method for regulating gene expression by using novel dCas9 |
| CN110885848A (en) * | 2019-08-19 | 2020-03-17 | 山东汇冠康博生物科技有限公司 | Method for improving protein expression level under stress condition |
| CN111197014A (en) * | 2020-04-01 | 2020-05-26 | 宜昌三峡普诺丁生物制药有限公司 | Corynebacterium glutamicum mutant strain and application thereof |
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| CN105441473A (en) * | 2014-08-18 | 2016-03-30 | 中粮营养健康研究院有限公司 | Glutamic acid producing strain and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110885848A (en) * | 2019-08-19 | 2020-03-17 | 山东汇冠康博生物科技有限公司 | Method for improving protein expression level under stress condition |
| CN110592126A (en) * | 2019-09-27 | 2019-12-20 | 北京理工大学 | Method for regulating gene expression at translation level by using rare codon |
| CN110607317A (en) * | 2019-09-27 | 2019-12-24 | 北京理工大学 | Method for regulating gene expression by using novel dCas9 |
| CN111197014A (en) * | 2020-04-01 | 2020-05-26 | 宜昌三峡普诺丁生物制药有限公司 | Corynebacterium glutamicum mutant strain and application thereof |
| CN111197014B (en) * | 2020-04-01 | 2022-11-08 | 宜昌三峡普诺丁生物制药有限公司 | Corynebacterium glutamicum mutant strain and application thereof |
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