CN105132388B - The carboxylase enzyme mutant R485P and its application that a kind of enzymatic activity improves - Google Patents
The carboxylase enzyme mutant R485P and its application that a kind of enzymatic activity improves Download PDFInfo
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- CN105132388B CN105132388B CN201510560217.2A CN201510560217A CN105132388B CN 105132388 B CN105132388 B CN 105132388B CN 201510560217 A CN201510560217 A CN 201510560217A CN 105132388 B CN105132388 B CN 105132388B
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- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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- C12Y604/00—Ligases forming carbon-carbon bonds (6.4)
- C12Y604/01—Ligases forming carbon-carbon bonds (6.4.1)
- C12Y604/01001—Pyruvate carboxylase (6.4.1.1)
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Abstract
The carboxylase enzyme mutant R485P improved the invention discloses a kind of enzymatic activity and its application, belong to genetic engineering and field of fermentation engineering.For the present invention by the R485 site mutations of the pyruvate carboxylase of Rhizopus oryzae into proline, obtained mutant enzyme activity improves 24.8%.Gene FUM1, while overexpression carboxylase enzyme mutant R485P are knocked out on the basis of PDC1 and ADH1 is knocked out, finds fumaric acid output increased 31.1%.Simultaneously by adding 32 μ g/L biotin, fumaric acid yield reaches 332 ± 12mg/L, 16.1% is improved compared with control group (290 ± 10.7mg/L).Invention efficient hardening carbon metabolism flow is entered the route of synthesis of fumaric acid by pyruvic acid, efficiently produces fumaric acid for structure Engineering Yeast and other dicarboxylic acids create condition, have good industrial application value and a prospect.
Description
Technical field
The carboxylase enzyme mutant R485P improved the present invention relates to a kind of enzymatic activity and its application, belong to hereditary work
Journey and field of fermentation engineering.
Background technology
Saccharomyces cerevisiae (Saccharomyces cerevisiae) is used as a kind of eucaryon pattern microorganism, because having：Heredity
Abundant information, it is Metabolically engineered easy to operate；Nutritional need is simple, and separation-extraction technology cost is cheap；At low ph conditions (even
pH<3.0) well-grown；It is resistant to the substrate of high concentration；It is GRAS (General Regarded As by FDA certifications
Safe) microorganism, fermented product have the advantages that security and (lactic acid, pyruvic acid, malic acid, prolonged as fermenting and producing carboxylic acid
Fumarate, butanedioic acid, α-ketoglutaric acid etc.) potential most suitable microorganism.However, saccharomyces cerevisiae is in high concentration sugar and the bar of ventilation
Batch fermentation can produce substantial amounts of ethanol under part, for using carboxylic acid, for the fermentation of target product, a large amount of accumulation of ethanol make
Carbon flow is obtained largely to lose.By the activity for weakening the key enzyme in ethanol pathway, it is possible to reduce towards the carbon metabolism flow of ethanol,
So as to reduce the loss of carbon flow；On this basis, can be by preventing or weakening the further metabolism of objective carboxylic acid, to build target
The route of synthesis of carboxylic acid.
The effect of pyruvate carboxylase is to convert pyruvic acid into oxaloacetic acid, and then carbon flow can be incorporated into objective carboxylic acid
Route of synthesis, therefore, the effect of pyruvate carboxylase can be vividly described as " biological valve ", how to strengthen carboxylase
Reaction, carbon flow is more effectively incorporated into the route of synthesis of objective carboxylic acid, turns into metabolic engineering saccharomyces cerevisiae production carboxylic acid
A key issue.There are some researches show the height of pyruvate carboxylase activity in cell to malic acid, butanedioic acid, glutamic acid
Accumulation important role.Any fermentation by saccharomyces cerevisiae technique using dicarboxylic acids as target product, it will all face same one
Individual problem：How to strengthen carboxylase reaction, promote carbon flow to flow to the route of synthesis of objective carboxylic acid by pyruvic acidHow to improve
The activity of pyruvate carboxylaseScheme provided by the present invention, have for the research using saccharomyces cerevisiae production carboxylic acid universal
Meaning.
The content of the invention
First purpose of the present invention is to provide a kind of carboxylase enzyme mutant, and the mutant is in amino acid sequence
On the basis of arranging parent's Rhizopus oryzae pyruvate carboxylase as shown in SEQ ID NO.1, the arginine mutation of the 485th is turned into
Proline.
In one embodiment of the invention, the nucleotides of the gene of parent's Rhizopus oryzae pyruvate carboxylase is encoded
Sequence is as shown in SEQ ID NO.2.
Second object of the present invention is to provide a kind of genetic engineering bacterium for expressing the mutant.
In one embodiment of the invention, the genetic engineering bacterium is using saccharomyces cerevisiae as host.
In one embodiment of the invention, the genetic engineering bacterium be with and meanwhile lacked pyruvate decarboxylase
PDC1, alcohol dehydrogenase ADH1, fumarase FUM1 saccharomyces cerevisiae are host.
In one embodiment of the invention, the nucleotide sequence such as Gene of the Pyruvate Decarboxylase Gene PDC1
ID:Shown in 850733, alcohol dehydrogenase gene ADH1 nucleotide sequence such as Gene ID:Shown in 854068, fumarase base
Because of FUM1 nucleotide sequence such as Gene ID:Shown in 855866.
Third object of the present invention is to provide a kind of engineering bacteria fermentation production two for utilizing and expressing the mutant
The method of first carboxylic acid.The dicarboxylic acids, including fumaric acid, malic acid, butanedioic acid, α-ketoglutaric acid etc..
In one embodiment of the invention, the dicarboxylic acids is fumaric acid.
In one embodiment of the invention, biotin is added during fermented and cultured.
In one embodiment of the invention, 32 μ g/L biotins are added during fermented and cultured.
In one embodiment of the invention, three gene deletion strains of carboxylase enzyme mutant will be overexpressed
Saccharomyces cerevisiae CEN.PK2-1C △ PDC1 △ ADH1 △ FUM1 seed liquor, is seeded to fermented and cultured
Cultivated under the conditions of base, with 28-32 DEG C, 150-250rpm.
In one embodiment of the invention, 24h genetic engineering bacterium seed will be cultivated under 30 DEG C, 220rpm with 5%
Inoculum concentration be transferred to fermented and cultured and be based on 30 DEG C, cultivate 96h under the conditions of 220rpm.
In one embodiment of the invention, fermentation medium contains：Without amino yeast nitrogen 3.4g/L, ammonium sulfate
5g/L, glucose 40g/L, leucine 100mg/L, tryptophan 20mg/L, histidine 20mg/L, uracil 20mg/L, calcium carbonate
5g/L。
The fumaric acid obtained the invention further relates to the mutant and the mutant is in food, feed, chemical industry, medicine
The application of thing preparation etc..
The mutant name of the present invention：It is on the basis of amino acid sequence shown in SEQ ID NO.1, using " original amino
Acid, amino acid sites, the amino acid replaced " represents mutant.For example R485P is represented the amino acid of position 485 by parent
Arginine (Arginine, R) replace with proline (Pro, P).
Beneficial effects of the present invention：(1) mutation of the arginine of the 485th of the pyruvate carboxylase of Rhizopus oryzae is turned into dried meat
Propylhomoserin, the specific enzyme activity of gained mutant improve 24.8% compared with parent；(2) carboxylase enzyme mutant is overexpressed by structure
Saccharomyces cerevisiae, the yield of dicarboxylic acids can be effectively improved, bar is created for efficiently production fumaric acid and other dicarboxylic acids
Part, there is good industrial application value and prospect；In pyruvate decarboxylase PDC1, alcohol dehydrogenase ADH1 and fumarase
The middle expression RoPYC for the saccharomycete that FUM1 is lacked simultaneously mutant R485P, fumaric acid output increased reached 312 ±
14mg/L, the RoPYC for relatively expressing wild type improve 31.1%；(3) by adding 32 μ g/L biotin, fumaric acid can be made
Yield is further increased to 332 ± 12mg/L, and 16.1% is improved compared with control group (290 ± 10.7mg/L).
Brief description of the drawings
Fig. 1：RoPYC-GFP albumen position observations；
Fig. 2：The fumaric acid yield comparison figure of various pyruvate carboxylase mutant strains；
Fig. 3：Influence of the RoPYC R485 site rite-directed mutagenesises to pyruvate carboxylase activity；
Fig. 4：The influence that different biotin additions accumulate to the fumaric acid for expressing RoPYC R485P engineering bacteria.
Embodiment
The assay method of ethanol, residual sugar content and fumaric acid：Detected using high performance liquid chromatograph (HPLC).Zymotic fluid
Through processing and supernatant is after 0.22 μm of filtering with microporous membrane, detects ethanol using RID (differential refraction detector) and residual sugar contains
Amount, fumaric acid content is detected using VWD (UV-detector), liquid-phase chromatography method is as follows：High performance liquid chromatograph is the U.S.
Waters Products, model 1515, chromatographic column are Aminex HPX-87H column (Bio-Rad).Column temperature：35℃；Stream
Dynamic phase：0.0275% (v/v) dilute sulfuric acid, through 0.22 μm of membrane filtration and degasification；Flow velocity：0.6mL/min；Detection time：
25min；Sample size：20μL.
The assay method (Bio-Rid nucleic acid instrument) of biomass：Dilute appropriate multiple with 0.1M HCl, set wavelength as
600nm, 200 μ L are taken to determine its light absorption value.
Seed culture medium：Glucose 2%, yeast extract 1%, peptone 2%, the deionization water capacity, pH is naturally, high pressure is gone out
Bacterium (115 DEG C, 20min).
Fermentation medium：Without amino yeast nitrogen 3.4g/L, ammonium sulfate 5g/L, glucose 40g/L, add respectively on request
Leucine 100mg/L, tryptophan 20mg/L, histidine 20mg/L, uracil 20mg/L, add calcium carbonate 5g/L, and liquid amount is
40mL/250mL.0-128 μ g/L biotin can be added.
Yeast conversion method (plasmid)：(1) the saccharomyces cerevisiae single bacterium of flat board activation is accessed in 3mL YPD fluid nutrient mediums
Fall, 30 DEG C, 220rpm overnight incubations；(2) EP pipe bacterium solutions are filled, room temperature 1min centrifugations are carried out under the conditions of 4000rpm；(3) fit
Sterilized water washing is measured, room temperature 1min centrifugations are carried out under the conditions of 4000rpm；(4) 1.0M LiAc 36 μ L, 10mg/ are sequentially added
The μ L of mL ssDNA 10 (ssDNA shifts to an earlier date boiling water bath 5min, places 5min on ice), plasmid 500ng, 50%PEG240 μ L, it is gentle mixed
It is even；(5) 42 DEG C of heat shocks 30 minutes；(6) room temperature 1min centrifugations are carried out under the conditions of 4000rpm, add 1mL sterilized waters to wash；(7)
4000rpm centrifuges 1min, stays suitable quantity of water pressure-vaccum cell, is coated with selective flat board, 30 DEG C of culture 3-5d.
Positioning of the embodiment 1RoPYC (pyruvate carboxylase in Rhizopus oryzae source) in saccharomyces cerevisiae
1st, promoter TEF1promoter and RoPYC genes ORF are cloned into pGFP33 carriers
According to homologous recombination kit principle, design both ends are with primer more than 15 bases homologous with carrier, such as table
1, lowercase is homology arm, and capitalization is PCR primer.
Table 1 expands the primer of RoPYC genes
Using pY15TEF1-RoPYC as template (Xu et al.Fumaric acid production in
Saccharomyces cerevisiae by silico aided metabolic engineering, 2012),
RoPYC-F, RoPYC-R (sequence is respectively as shown in SEQ ID NO.3, SEQ ID NO.4) be primer amplify TEF1p and
RoPYC ORF frames (obtained PCR primer contains encoding amino acid sequence SEQ ID NO.1 nucleotide sequence), amplification is produced
Thing is connected to pGFP33 carriers, converts large intestine competent cell, LB flat board of the coating added with ampicillin.Bacterium colony PCR is tested
Demonstrate,prove the transformant grown and propose plasmid enzyme restriction checking, correct transformant is carried out protecting bacterium and is sequenced, plasmid designations are named as
pGFP33-RoPYC。
2nd, expression of the RoPYC in saccharomyces cerevisiae
Build three gene deletion strains Saccharomyces cerevisiae CEN.PK2-1C △ PDC1 △ ADH1 △
FUM1 (construction method refers to the patent application of Application No. 201410340560.1), recombinant plasmid pGFP33-RoPYC is turned
Change to three gene deletion strains Saccharomyces cerevisiae CEN.PK2-1C △ PDC1 △ ADH1 △ FUM1, conversion
Method is shown in embodiment.
3rd, RoPYC is positioned
The genetic engineering bacterium colony inoculation of activation is taken from SD-Ura flat boards in 3mL SD-Ura fluid nutrient mediums, 30
DEG C, 220rpm overnight incubations.The μ L of bacterium solution 500 are taken to access in new 4.5mL SD-Ura fluid nutrient mediums.4h is cultivated, is made at cell
In exponential phase.Take 1mL bacterium solutions to pour into 1.5mL EP pipes, add 1 μ L DAPI dye liquors, fully mix, lucifuge dyes on ice
10min.4000rpm centrifuges 1min, discards part supernatant.Take appropriate sectioning cells, pine and cypress oil, is shown with Nikon80i fluorescence in drop
Micro mirror is observed.100 times of amplification, yield value 2.0, time for exposure 1s, fluoroscopic examination is carried out, takes DIC and green fluorescence, blueness
Photo under fluorescence excitation.Each bacterial strain randomly selects 3 transformants, with 100 × object lens of fluorescence microscope observe the DIC visuals field,
Cellular morphology under green glow and blue light, as a result as shown in Figure 1, the results showed that the electrodes method is in the kytoplasm of brewing yeast cell.
Embodiment 2RoPYC rite-directed mutagenesises and expression
Rite-directed mutagenesis is carried out with PCR method, saturation mutation is carried out to RoPYC R485 sites, by the smart ammonia in the 485th site
Acid mutation is into other 19 amino acid.Be primer using the pY15TEF1-RoPYC plasmids F as template, containing mutational site and R,
Takara companies high-fidelity enzyme PrimeSTAR GXL enter performing PCR and amplify whole plasmid.Digestion system includes 1 μ L PCR primers
And 1 μ L Dpn I enzyme, the μ L of cumulative volume 20,37 DEG C of digestions are stayed overnight.Digestion products carry out fragment purification.The μ L of purified product 5 are taken to turn
Change 30 μ L competent cell Trans1-T1, be coated with LA flat boards, the transformant inoculation LA culture mediums grown, upgrading grain sends to Shanghai
Raw work sequencing.
Wherein, (sequence is respectively such as SEQ ID NO.5, SEQ ID by the primers F (Pro) for R485P mutation, R (Pro)
Shown in NO.6), as shown in table 2.
The rite-directed mutagenesis primer of table 2
Note：Italic underline for mutating alkali yl, amino acid are on right side corresponding to them.
Correct mutant is sequenced in selection, in three gene deletion strains Saccharomyces cerevisiae
The mutant is overexpressed in CEN.PK2-1C △ PDC1 △ ADH1 △ FUM1, has obtained series of genes engineering bacteria.By gene work
Journey bacteria strain, with the conditions of, fermenting experiment is carried out, has compared the amino acid in the R485 sites of the PYC in Rhizopus oryzae
Saturation mutation produces the influence of fumaric acid to saccharomyces cerevisiae, as a result as shown in Fig. 2 result shows the production of fermenting and producing fumaric acid
Amount, wherein expression R485P engineering bacteria yield has reached 312 ± 14mg/L, relatively expresses wild type up to more than 200mg/L
RoPYC improves 31.1%.
The mutant R485P of embodiment 3 expression and the production of fumaric acid
Compare the specific enzyme activity of carboxylase enzyme mutant R485P and parent enzyme, and different biotin additions pair
The influence of the fumaric acid accumulation of R485P genetic engineering bacterium is expressed, as a result as shown in Figure 3 and Figure 4.
Condition of culture：The genetic engineering bacterium seed that 24h is cultivated under 30 DEG C, 220rpm is transferred to fermentation with 5% inoculum concentration
Culture based on 30 DEG C, cultivate 96h under the conditions of 220rpm.
As seen from Figure 3, mutant R485P enzyme activity increase, adds 24.8% than parent respectively.
As seen from Figure 4, the biotin for adding various concentrations is advantageous to the accumulation of fumaric acid, when addition wild Oryza species
When middle biotin concentration is 32 μ g/L, fumaric acid yield reaches 332 ± 12mg/L, the control group (290 with not adding biotin
± 10.7mg/L) compared to improving 16.1%.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection model of the present invention
Enclose being defined of being defined by claims.
Claims (9)
1. a kind of carboxylase enzyme mutant, it is characterised in that the mutant is in amino acid sequence such as SEQ ID NO.1
On the basis of shown Rhizopus oryzae pyruvate carboxylase, the arginine mutation of the 485th is turned into proline.
2. encode the gene of mutant described in claim 1.
3. carry the carrier or genetic engineering bacterium of gene described in claim 2.
4. the carrier or genetic engineering bacterium described in claim 3 are in the related food of dicarboxylic acids, feed, chemical industry, medicine preparation
The application of aspect.
5. the method for fumaric acid is produced using mutant described in claim 1.
A kind of 6. method for promoting fumaric acid accumulation using mutant described in claim 1, it is characterised in that in encoding pyruvate
In the saccharomycete that acid decarboxylase PDC1, alcohol dehydrogenase ADH1 and fumarase FUM1 gene lack simultaneously, it is overexpressed and compiles
The gene of the code carboxylase enzyme mutant.
7. according to the method for claim 6, it is characterised in that the nucleotide sequence of the Pyruvate Decarboxylase Gene PDC1
Such as Gene ID:Shown in 850733, alcohol dehydrogenase gene ADH1 nucleotide sequence such as Gene ID:Shown in 854068, prolong recklessly
Rope phytase gene FUM1 nucleotide sequence such as Gene ID:Shown in 855866.
8. according to the method for claim 6, it is characterised in that biotin is added during fermented and cultured.
9. according to the method for claim 6, it is characterised in that lack three genes for being overexpressed carboxylase enzyme mutant
Bacterial strain Saccharomyces cerevisiae CEN.PK2-1C △ PDC1 △ ADH1 △ FUM1 seed liquor is lost, is seeded to hair
Ferment culture medium, cultivated under the conditions of 28-32 DEG C, 150-250rpm.
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| CN105754963A (en) * | 2016-05-19 | 2016-07-13 | 江南大学 | Method for improving yield of fumaric acid |
| CN109609486B (en) * | 2019-01-30 | 2020-07-24 | 浙江华睿生物技术有限公司 | Fumarase mutant and application thereof |
Citations (2)
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|---|---|---|---|---|
| WO1995006114A1 (en) * | 1993-08-24 | 1995-03-02 | Ajinomoto Co., Inc. | Variant phosphoenolpyruvate carboxylase, gene thereof, and process for producing amino acid |
| CN104099258A (en) * | 2014-07-16 | 2014-10-15 | 江南大学 | Saccharomyces cerevisiae genetically-engineered bacterium capable of realizing ethanol accumulation reduction and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995006114A1 (en) * | 1993-08-24 | 1995-03-02 | Ajinomoto Co., Inc. | Variant phosphoenolpyruvate carboxylase, gene thereof, and process for producing amino acid |
| CN104099258A (en) * | 2014-07-16 | 2014-10-15 | 江南大学 | Saccharomyces cerevisiae genetically-engineered bacterium capable of realizing ethanol accumulation reduction and application thereof |
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| ADG65259.1;Xu,G.等;《GenBank》;20130115;第1-2页 * |
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