CN107723302A - A kind of method for being overexpressed Candida glycerolgenesis CgGAD1 and improving osmotic pressure tolerance - Google Patents
A kind of method for being overexpressed Candida glycerolgenesis CgGAD1 and improving osmotic pressure tolerance Download PDFInfo
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Abstract
The present invention relates to the clone of hypertonicity-resistant functional gene CgGAD1 from Candida glycerolgenesis (Candida glycerinogenes) a kind of and its application, belong to molecular biology, biological technical field.Method is, from Candida glycerolgenesis C.glycerinogenes DNA sequence DNA amplification obtain gamma aminobutyric acid synthesis key gene CgGAD1, full length gene 1830bp, intronless.The gene is hypertonicity-resistant functional gene, and overexpression CgGAD1 can increase accumulation of the gamma aminobutyric acid in intracellular, improves growth of the bacterial strain under the conditions of hypertonic.Methods of the C.glycerinogenes to osmotic pressure tolerance is further improved using genetic engineering means the invention provides a kind of, excellent genetic material and Research Thinking are provided for the improvement of the resistance of other microorganisms and higher eucaryote.
Description
Technical field
The present invention relates to a kind of clone of Candida glycerolgenesis CgGAD1 genes, overexpression and osmophilic strain salt-resistance
Functional analysis and application, belong to molecular biology and biological technical field.
Background technology
Candida glycerolgenesis (Candida glycerolgenesis) is one plant of tool that China possesses independent intellectual property right
There is the yeast strain of excellent fermenting property, be applied to the industrialized production of glycerine for a long time.C.glycerolgenesis has height
The characteristics of yield, high conversion, big production intensity.The conventional pattern Yeast Phase ratio with S.cerevisiae etc.,
C.glycerolgenesis has higher osmotic pressure tolerance, can be carried out in the culture medium more than 550g/L glucose
Normal physiological metabolism, therefore there is potential application prospect in the fields such as thick mash fermentation.
Yeast generally can all meet with the ring of various outsides in growth or breeding especially under industrial fermentation environment
Border pressure.Especially outside hyperosmosis frequently can lead to cell interior water loss, cause the loss of cell expansion pressure, from
And causing cellular atrophy, this performance to yeast physiological function is totally unfavorable.In order to tackle the ambient exterior atmospheric pressure of complexity,
Yeast cells has gradually formed a series of specific Adaptive mechanisms during long-term evolution, can accurately experience surrounding
The change of environment simultaneously responded rapidly by different regulation mechanisms, and cell is protected with this.In S.cerevisiae, carefully
Born of the same parents mainly provide osmotic pressure protection by using the intracellular glycerol Rapid Accumulation mechanism of Hog1 mediations.In addition also exist more
Kind regulation mechanism, such as the stable state (Na driven dependent on ATP inside ion+、K+Active transport and H+Antiport), TOR
(Cell wall integrity, cell membrane are complete by (Target of rapamycin, rapamycin target signal pathway) and CWI
Whole sexual approach) etc..S.cerevisiae is also by accumulating cation, sugared (such as trehalose, sucrose, glycogen), polyalcohol (such as sweet dew
Alcohol, sorbierite etc.) and the compatible material such as amino acid (such as proline, arginine and glutamic acid) recover the turgor pressure of cell.
This kind of material had both been probably derived from internal synthesis, it is also possible to absorbs from external environment, they are the osmotic pressure response regulation and control of yeast
The important composition of mechanism.Under the conditions of hyperosmosis, C.glycerolgenesis γ-aminobutyric acid permease gene by
Strong induction, its intracellular content obtain a certain degree of accumulation, and external source addition γ-aminobutyric acid can promote in the medium
Growths of the C.glycerolgenesis under the conditions of osmotic pressure, show that C.glycerolgenesis can utilize γ-aminobutyric acid
As osmoprotectant.
The content of the invention
It is an object of the invention to provide a kind of γ-ammonia for deriving from Candida glycerolgenesis (C.glycerolgenesis)
Base butyric acid synthesizes key enzyme glutamic acid decarboxylase gene CgGAD1, and the gene nucleotide series are as shown in SEQ ID NO.1.
It is a further object of the present invention to provide a kind of side for improving Candida glycerolgenesis thalli growth under the conditions of hypertonic
Method.The CgGAD1 of itself is derived from by overexpression, increases accumulation of the γ-aminobutyric acid in intracellular, promotes bacterial strain hypertonic
Under the conditions of thalli growth.
Technical scheme：A kind of method for improving Candida glycerolgenesis and resisting extraneous hyperosmosis, utilizes urine
The glycerine industrial producing strain Candida glycerolgenesis C.glycerolgenesis U5 of pyrimidine auxotrophic are research object,
Clone obtains the glutamic acid decarboxylase gene CgGAD1 from Candida glycerolgenesis, and it is crossed into table in the method for integrant expression
Up in C.glycerolgenesis U5, the intracellular alpha-aminobutyric acid content of recombinant bacterium is significantly raised, and it is hypertonic
The thalli growth under the conditions of pressure also significantly improves thoroughly, shows effectively to protect by overexpression glutamic acid decarboxylase gene
Candida glycerolgenesis resists extraneous infiltration pressure stress.
Beneficial effects of the present invention：The present invention separates from Candida glycerinogenes DNA sequence
To γ-aminobutyric acid synthesis key enzyme glutamic acid decarboxylase gene GAD1.Gene order total length 1830bp, intronless, compile
609 amino acid of code.Result of study shows the overexpression GAD1 genes in Candida glycerolgenesis, can improve intracellular γ-ammonia
The content of base butyric acid, and then promote growth of the bacterial strain under the conditions of hyperosmosis.The result of study finds that γ-aminobutyric acid can be made
For osmoprotectant in intracellular accumulation to protect Candida glycerolgenesis to resist extraneous osmotic pressure.GAD1 genes are expanded
The genetic resources of microorganism impermeabilisation pressure stress research, is also provided for the resistance to hypertonic physiological Mechanism research of Candida glycerolgenesis
Thinking.To better profit from and transforming Candida glycerolgenesis from now on, to realize its application in the fields such as thick mash fermentation
Research foundation is provided.
Brief description of the drawings:
The C.glycerinogenes GAD1 genetic fragments that Fig. 1 .PCR amplifications obtain.
M:DL2000Marker；1:The fragment obtained with primer GAD-F and GAD-R amplification.
Fig. 2 .C.glycerinogenes U5 and C.glycerinogenes U5/pGAD1 are in the solid containing 1M NaCl
Upgrowth situation on culture medium.
Fig. 3 .C.glycerinogenes U5 and C.glycerinogenes U5/pGAD1 are containing different NaCl concentrations
Fluid nutrient medium in thalli growth.
Embodiment
The present invention is expanded on further below by specific embodiment, these embodiments are merely to illustrate the present invention and do not had to
In the limitation present invention.
Embodiment 1：The clone of C.glycerinogenes CgGAD1 genes
In embodiment, the culture to bacterial strain uses YEPD culture mediums (g/L) respectively：Glucose 20, peptone 20, yeast is taken out
Extract 10, agar 20 (solid medium), pH5.5, and minimal medium (g/L)：Glucose 20, no amino yeast nitrogen 6.7,
Agar 20 (solid medium), pH5.5.C.glycerinogenes is cultivated in YEPD culture mediums at 30 DEG C to logarithmic growth
Phase, thalline is collected, genomic DNA is extracted with glass bead method.Utilize primer GAD-F:5′-
CGCGGATCCATGACACTTTCCAGCCATGT-3′；GAD-R:5′-CTTGGTACCTTAACA AATAA
CTTTTGCATTTGAG-3 ', enter performing PCR amplification by template of genomic DNA, as a result as shown in Figure 1.The purpose obtained will be expanded
Fragment is connected on pMD-19T carriers, is converted to e. coli jm109, enterprising in the LB solid plates containing ampicillin
Row screening, picking transformant carry out bacterium colony PCR screening positive clones, and are passed to Shanghai life work and are sequenced.Sequencing result
Nucleotide sequence total length 1830bp is shown, sequence refers to SEQ ID NO.1, is named as CgGAD1.
Embodiment 2：CgGAD1 is overexpressed recombinant bacterium U5/pGAD1 structure
The gene amplification fragment and integrant expression obtained in embodiment is carried respectively using Restriction Enzyme BamH I and Kpn I
Body pURGAP carries out double digestion respectively, and the fragment of acquisition connects overnight through T4-DNA ligases at 16 DEG C, is transformed into E.coli
In JM109 competence, transformant is obtained on amicillin resistance flat board, positive transformant is screened using bacterium colony PCR and incites somebody to action
It is forwarded in LB culture mediums, and the integrant expression plasmid pGAD1 that plasmid extraction obtains CgGAD1 genes is carried out after being incubated overnight.Weight
Group plasmid carries out linearized enzyme digestion with restriction enzyme Hind III, and C.glycerinogenes urine is transferred to LiAc methods
In pyrimidine auxotrophic mutant strain U5.Transformant is obtained after being cultivated 3 days on minimal medium, bacterium colony PCR screenings obtain
The recombinant bacterium U5/pGAD1 of CgGAD1 gene overexpressions.
Embodiment 3：It is overexpressed the application that CgGAD1 improves C.glycerinogenes osmotic pressure tolerances
1st, it is overexpressed upgrowth situation of the CgGAD1 recombinant bacterium on the solid medium containing 1M NaCl.
Recombinant bacterium U5/pGAD1 and control strain U5 are cultivated to exponential phase respectively, take the thalline of equivalent respectively with nothing
Bacterium physiological saline prepares bacteria suspension and serial dilution.Draw 3 μ l bacterium solutions respectively with liquid-transfering gun, put down according to the concentration gradient after dilution
Row is dropped on the solid medium containing 1M NaCl；Static gas wave refrigerator in 30 DEG C of constant incubators is transferred to after drop absorption
24h, colony growth situation is observed, as a result sees Fig. 2, show that being overexpressed CgGAD1 improves life of the bacterial strain under the conditions of osmotic pressure
It is long.
2nd, it is overexpressed thalli growth of the CgGAD1 recombinant bacterium in the fluid nutrient medium containing different NaCl concentrations.
Recombinant bacterium U5/pGAD1 and control strain U5 are switched to the YEPD Liquid Cultures containing various concentrations NaCl
In base, 30 DEG C, in 200rpm shaking table culture, using the light absorption value at spectrophotometric determination 600nm, as a result see to stationary phase
Fig. 3.Overexpression CgGAD1 is remarkably improved growth ability of the bacterial strain under the conditions of hypertonic.
Sequence table
<110>Southern Yangtze University
<120>A kind of method for being overexpressed Candida glycerolgenesis CgGAD1 and improving osmotic pressure tolerance
<130> 20171128
<141> 2017-11-30
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1830
<212> DNA
<213>Candida glycerolgenesis (Candida glycerolgenes)
<400> 1
atgacacttt ccagccatgt tgacacagat gctttagagg ataagatttt tgagaagtct 60
tctccaaaac acaagttact ccaaaagtac aatgacatac taagaagaca cgggaacgac 120
gagactaatt ttgaggaaac acccgaagct gcagttgcaa gattccgtgg agtagcaaac 180
aagtatagaa ttccagaaac tggaatgccg tcagacctag catacaatat tgttcatgac 240
gaaatggcat tagatgggtc aacaactcta aatttggcat cttttgtcaa tgtccacacc 300
gatgaggaga ccatgaagtt gattacccaa aacttaacca aaaatttggc tgacaatgac 360
gaatatccaa tgttaattga gatgcaagag cgttgtattt cgattcttgc aaatctgtgg 420
catgcgccat tagttaccga agactctggt attaagaccc cccatggtaa agaggacatc 480
tcaactttca agagaagagc gattggtaca ccatgtacag gctcatccga aggtgtcatg 540
ttaggtgggt tggccatgaa aaaaaattgg caagcaaaga gaaaggccaa gggtttgtct 600
acggataaac caaatattct gatggcttct tgtgcacaag ttgcattaga aaaattcgca 660
agatattttg atgttgaaaa cagattgatt ggtgtgtctg ataaggactt tttgattgat 720
gttgacaaaa ttagagagaa tttggatgag aatacaattg gtatctatgt tattgttggt 780
tcaacataca ccggtggttt tgaaaacgtt gagaaaattg ctgaaatctt ggatcagtac 840
gaaaaggaaa caggacattg gattccaatc cacgttgatg ccgcatctgg aggttttatt 900
gctccaatca tctatccgga attcaattgg gatttccaaa ttccaagggt tatgtcgatc 960
tctacatcgg gtcacaagtt tggtctagtt actgtgggtt taggctgggt gttgttcaga 1020
gatgaaagtt ggttaccaaa gagcttaaga tttgagttat cttaccttgg tggcttggag 1080
gaatctttta gcttgaattt ttccagacct ggttatcaaa ttgttcacca atactataat 1140
ttcctaagat ggggtaaaca aggatactac gatgtctttg ataatgcatt aactaatgca 1200
agattgttat cattgttctt ggaagaatct ggttacttta cttgtgtgtc taatttacat 1260
ttacctttag gtatgagtgc aagaaacaga gatccaagtt gggagccaag tcaggcaaac 1320
gagattatag atgagcacga caaattcaac ccagcattac cggttgtttc attccaactc 1380
actaaagaat tttcagaaga gtatcccgaa atacctcagt ctttaatctc tacctctttg 1440
agaaagaaga agtggattat accaaactac ccattgccaa gaattaacgt tccaaaggaa 1500
aatgaggatg gtgaagtcga agacgatgaa agtttgtgga acgaagctaa tgggttaaac 1560
aacgaaattt taagagttgt tgtcaagtac aacttaactg cccagttgct agataaatta 1620
atgcatgata taattgatgt tgttgaaggt cttatcaaat cagttaagtt ggtcagaaaa 1680
aatatcagcg aatcgaaaac tagtaccgat aagcacaatg aggaactaat ttacaatatg 1740
ttgctatcta tttctaatga tggtgatgaa cgtttgatta agttgaagac tgatgaaaac 1800
aagacctcaa atgcaaaagt tatttgttaa 1830
Claims (2)
1. a kind of glutamic acid decarboxylase gene CgGAD1 from Candida glycerolgenesis, its nucleotide sequence such as SEQ ID
NO:Shown in 1.
2. the function of the nucleotide sequence according to claim 1, it is characterised in that Candida glycerolgenesis can be significantly improved
Hyperosmosis tolerance.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114231429A (en) * | 2021-12-09 | 2022-03-25 | 江南大学 | Recombinant bacterium for expressing candida glycerinogenes RNA helicase and application thereof |
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|---|---|---|---|---|
| CN103131718A (en) * | 2012-12-28 | 2013-06-05 | 江南大学 | Cloning of hypertonicity-resistant functional gene CgHog1 from Candida glycerinogenes and application thereof |
| CN104830745A (en) * | 2015-04-28 | 2015-08-12 | 江南大学 | Efficient production method of gamma-aminobutyric acid |
| JP2015192620A (en) * | 2014-03-31 | 2015-11-05 | 栃木県 | Method for producing gaba using malt root |
| CN105400813A (en) * | 2015-12-28 | 2016-03-16 | 江南大学 | Pichia pastoris gene engineering bacteria for recombinant expression of human glutamic acid decarboxylase |
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2017
- 2017-11-30 CN CN201711232385.4A patent/CN107723302B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103131718A (en) * | 2012-12-28 | 2013-06-05 | 江南大学 | Cloning of hypertonicity-resistant functional gene CgHog1 from Candida glycerinogenes and application thereof |
| JP2015192620A (en) * | 2014-03-31 | 2015-11-05 | 栃木県 | Method for producing gaba using malt root |
| CN104830745A (en) * | 2015-04-28 | 2015-08-12 | 江南大学 | Efficient production method of gamma-aminobutyric acid |
| CN105400813A (en) * | 2015-12-28 | 2016-03-16 | 江南大学 | Pichia pastoris gene engineering bacteria for recombinant expression of human glutamic acid decarboxylase |
Non-Patent Citations (1)
| Title |
|---|
| JI H等: "Identification of a novel HOG1 homologue from an industrial glycerol producer Candida glycerinogenes", 《CURR MICROBIOL.》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114231429A (en) * | 2021-12-09 | 2022-03-25 | 江南大学 | Recombinant bacterium for expressing candida glycerinogenes RNA helicase and application thereof |
| CN114231429B (en) * | 2021-12-09 | 2023-07-25 | 江南大学 | Recombinant bacterium for expressing candida glycerinogenes RNA helicase and application thereof |
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